Potassium (K+) channels are major determinants of cell excitability and play crucial roles in many physiological processes. Central to a mechanistic understanding of ion channels is the process of gating, the opening and closing of the pore to ionic flow. Gating malfunctions can have disastrous physiological consequences. The overall objective of this grant is to understand the molecular mechanisms of ligand modulation and gating in K+ channels by employing functional, structural, and theoretical analysis on model prokaryotic K+ channels, homologues of eukaryotic K+ channels. Bacterial channels lend themselves to the biochemical and structural studies necessary to investigate these mechanisms. Our first major aim is to understand ligand gating in K+ channels using KcsA as a model. In Aims 1.1 and 1.2 we propose to determine directly the pKas (the intrinsic proton binding affinities) of two proposed proton-sensing residues using NMR spectroscopy, in both open and closed states. We will take advantage of mutants with changed pH gating as determined with electrophysiology to guide interpretations of peak assignments and chemical shift changes in the NMR spectra. We will also use crosslinked closed and open KcsA constructs that will allow us to determine pKas of pH sensing residues in the absence of the conformational change that gates the channel. To our knowledge, this would be the first direct determination of intrinsic ligand binding affinities to both open and closed states. In Aim 1.3 we will formulate a Monod-Wyman-Changeux model for proton gating in KcsA constrained with results from the combined approach of modeling, mutagenesis, electrophysiology, X-ray crystallography and NMR. X-ray crystallography will be used to structurally determine the effect of channel mutations. Our second major aim is to understand gating of Ca2+-activated K+ channels using MthK as a model. Aims 2.1 and 2.2 investigate the physical location of the gate modulated by Ca2+. We will investigate whether the MthK Ca2+ activation gate is at the bundle crossing or at the selectivity filter by determining if the access of intracellular blockers that bnd inside the vestibule of K+ channels (between the two gates) depends on whether the channel is open or closed. Accessibility of the blockers to the vestibule in the absence of Ca2+ is an indication of a selectivity filter gate. In Aim 2.3 we propose to investigate MthK slow desensitization, a phenomenon observed only in liposomes, not in bilayer recordings, by altering the bilayer composition. This phenomenon is intriguing as its differential occurrence would indicate that a fundamental, physiologically relevant, channel property is digitally modulated by bilayer parameters. We will use a stopped-flow bulk assay that measures the quenching of a fluorophore upon Tl+ entry through MthK-reconstituted vesicles to determine the affinity and the kinetics of blockers both in the presence and absence of Ca2+ as well as determine the effect of lipids on desensitization. The proposed aims will provide new insights into the fundamental K+ channel properties, which will be readily applicable to our understanding of the broader family of eukaryotic K+ channels.